Purification systems using glass or metal tubes which contain a packed column of separation medium, for example, beads or particles, are known. These tubes are known as column boxes. Because the separation medium is compacted within the column boxes, the flow rates are slow and the column boxes have a limited capacity. Therefore, prior art purification technology has focused on increasing the porosity of the separation medium to increase the flow rates and capacity within the column box. The object of these known systems is to purify the largest amount of material within the shortest amount of time while keeping the amount of contaminants low and the product yields high. One problem with the old purification technology is that increasing porosity of the separation medium achieved faster flow rates and capacity, but reduced product yields and quality.
A past method, which does not use a column box, utilized a dynamic (i.e., mechanical forces at the site of filtration) filtration system with a variety of separation medium. This old method and apparatus also failed in part because the sheering forces of the dynamic system damaged the separation medium and the bio-compounds being purified.
Prior methods of affinity separation involving dynamic filtration of cellulose and non-cellulose beads have the disadvantage that the beads degrade due to sheering forces inherent in dynamic filtration. These sheering forces irreversibly damage bead supports and the sheer-sensitive biological molecules grafted or bound to them. Generally, many porous beads will fragment due to the agitation and sheering forces contained in the dynamic affinity separation apparatus. The present invention overcomes the disadvantages of the prior art by utilizing a static affinity separation method which is gentle on beads and biological molecules, but causes intermixing of the target compound to be purified with the sheer-sensitive beads and biological molecules which flow through the static tangential flow filters.
The prior art discloses the use of polystyrene beads in dynamic filtration apparatuses for affinity separation of biological compounds. Certain disadvantages are involved in the use of polystyrene beads, such as high non-specific adsorbing of biological molecules on their hydrophobic surfaces. Further, polystyrene beads have open pores on the surface of the bead which entrap contaminants which will co-purify with wanted products and decrease yield and purity of a target compound. Polystyrene has been found to exhibit a high degree of agglomerate and to adhere to the filters used in dynamic affinity separation methods. This agglomeration of the polystyrene beads allows for debris to become trapped and spoils the filtration affinity system by clogging the filter. A preferred embodiment of the present invention overcomes the disadvantages of the prior art by adding a multitude of linkers to the bead surface to increase bead coating. Bead coating reduces unwanted agglomeration and filter clogging. This multitude of linkers reduces agglomeration and non-specific binding, resulting in increased stability and reduced entrapment of unwanted contaminants, while enabling the attachment of the target compounds desired to be separated.
The prior art discloses the use of cellulose in dynamic filtration apparatuses for affinity separation of biological compounds. The prior art cellulose particles available are highly porous particles which exhibit entrapment of target and contaminants. Moreover, these highly porous prior art cellulose particles can swell from 25% to 400% of their original size in aqueous medium. Additionally, the prior art cellulose particles are highly sensitive to sheering forces, thus resulting in fragmentation in a dynamic filter. The present invention uses non-porous cellulose beads in a static tangential flow system to prevent bead fragmentation.